The Molten Globule of ¦Â₂-Microglobulin Accumulated at pH 4 and Its Role in Protein Folding

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• 作者：Atsushi Mukaiyama¹ ; ^&dagger ; ; Takashi Nakamura¹ ; ^&dagger ; ; Koki Makabe¹ ; ² ; ¹ ; Kosuke Maki¹ ; ² ; ¹ ; Yuji Goto³ ; Kunihiro Kuwajima¹ ; ² ; ^{kuwajima@ims.ac.jp}
• 关键词：¦Â2m ; ¦Â2-microglobulin ; MG ; molten globule ; GFP ; green fluorescent protein ; PFG ; pulse field gradient ; 1D ; one-dimensional ; 2D ; two-dimensional ; HSQC ; heteronuclear single quantum coherence ; HMQC ; heteronuclear multiple quantum coherence ; PDB ; Protein Da
• 刊名：Journal of Molecular Biology
• 出版年：2013
• 出版时间：23 January, 2013
• 年：2013
• 卷：425
• 期：2
• 页码：273-291
• 全文大小：1024 K

文摘

The acid transition of ¦Â₂-microglobulin (¦Â2m) was studied by tryptophan fluorescence, peptide circular dichroism, and NMR spectroscopy. The protein exhibits a three-state transition with an equilibrium intermediate accumulated at pH 4 (25 ¡ãC). The pH 4 intermediate has typical characteristics of the molten globule (MG) state; it showed a native-like secondary structure without specific side-chain tertiary structure, and the hydrodynamic radius determined by pulse field gradient NMR was only 20 % larger than that of the native state. The accumulation of the pH 4 intermediate is very analogous to the behavior of apomyoglobin, for which the pH 4 MG has been well characterized, although ¦Â2m, a ¦Â-protein, is structurally very different from ¦Á-helical apomyoglobin. NMR pH titration of histidine residues of ¦Â2m has also indicated that His84 has an abnormally low pK_a value in the native state. From the pH dependence of the unfolding transition, the protonations of this histidine and 10 weakly abnormal carboxylates triggered the transition from the native to the MG state. This behavior is again analogous to that of apomyoglobin, suggesting a common mechanism of production of the pH 4 MG. In contrast to the folding of apomyoglobin, in which the MG was equivalent to the burst-phase kinetic folding intermediate, the burst-phase refolding intermediate of ¦Â2m, detected by stopped-flow circular dichroism, was significantly more structured than the pH 4 intermediate. It is proposed that the folding of ¦Â2m from its acid-denatured state takes place in the following order: denatured state ¡ú MG ¡ú burst-phase intermediate ¡ú native state.